The overall goal of this application is to elucidate the function of Ena/VASP phosphorylation by protein kinase A (PKA) in axon outgrowth and guidance in the developing central nervous system. First, Ena/VASP-null mice will be generated. If it is not possible to create triple knock-out mice due to early prenatal lethality, a conditional knock-out strategy will be employed by means of the Cre/lox system of recombination. Second, phosphorylation mutants of Ena/VASP proteins will be introduced into Ena/VASP-null neurons and their effects on growth cone motility and axon outgrowth will be assessed. To test the function of Ena/VASP phosphorylation on axon guidance cortical neurons containing phosphorylation mutants will be exposed to gradients of netrin and BDNF; two molecules known to signal through PKA. Ena/VASP proteins have also been implicated in binding A kinase anchoring proteins (AKAPs). In order to determine which AKAPs associate with Ena/VASP proteins in cortical neurons co-immunoprecipitations and gel overlays will be performed with the type II regulatory subunit of PKA. AKAPs discovered to bind Ena/VASP proteins will be cloned and labeled with YFP. Fluorescent resonance energy transfer (FRET) will be performed with CFP-Ena/VASP proteins to determine where and when Ena/VASP proteins and AKAPs interact in growth cones. An elucidation of the function of Ena/VASP proteins in CNS development will be important for understanding human CNS diseases where directed neuronal migration and outgrowth are impaired. [unreadable] [unreadable]